Mahood - Hama-Salih: Characterization of genetic diversity and relationship in almond genotypes by RAPD and ISSR markers in Sulaimani governorate - 1739 - CHARACTERIZATION OF GENETIC DIVERSITY AND RELATIONSHIP IN ALMOND (PRUNUS DULCIS [MILL.] D.A. WEBB.) GENOTYPES BY RAPD AND ISSR MARKERS IN SULAIMANI GOVERNORATE MAHOOD, A. M. R.* – HAMA-SALIH, F. M. Horticulture Department, College of Agricultural engineering Sciences, University of Sulaimani Sulaimani-Kurdistan Region, Iraq *Corresponding author e-mail: [email protected]; phone: +96-4770-1532-547 (Received 13th Oct 2019; accepted 21st Jan 2020) Abstract. Genetic diversity of 38 almond local genotypes was investigated using RADP and ISSR markers with the analysis of nut morphology. Samples were taken from five locations for this study, including Sharbazher, Mergapan, Qaradakh, Barznja, and Hawraman. Almond nuts width, length and thickness were studied and their mean values were observed to range between 16.18-27.21 mm, 24.18-41.07 mm and 11.49-16.81 mm, respectively. Polymorphic bands of mean values were 9.5 for random amplified polymorphic DNA (RAPD) and 8 for inter-simple sequence repeat (ISSR). The PIC values were recorded for RAPD primes to range between 0.77 to 0.97 and those for ISSR primers were also verified between 0.36 to 0.97. Based on Jaccard similarity coefficients, the genetic distances were recorded between 0.32 (B-G3 vs. B-G4) (M-G2 vs. M-G1) to 0.75 (H-G5 vs. Q-G1) and all genotypes were grouped into 3 major clusters (A, B and C) with a mean dissimilarity 0.535 for 20 RADP markers. In the case of the 15 ISSR markers, a genetic distance between 0.19 (H-G13 vs. H-G12) to 0.78 (H-G5 vs. B-G6) was also observed, with four clades (A, B, C and D) with a mean dissimilarity of 0.485. According to STRUCTURE analysis, all genotypes were divided into two groups. Analysis of molecular variance (AMOVA) demonstrated a high-level genetic differentiation within a population 88% for RAPD and 87% for ISSR. Keywords: morphological traits, genetic relationship, genetic structure, random amplified polymorphic DNA, inter-simple sequence repeat Introduction Almond (Prunus dulcis (Mill.)) D.A. Webb syn. P. amygdalus L. Batsch is a commercially important fruit plant, from the Rosaceae family (Zhu, 2014; Sakar et al., 2019). The place of wild almond is originated at the arid mountainous region of Central Asia and deserts of western China, Iran, Turkistan, Afghanistan, Kurdistan, and Southwest Asia with subsequent expansion into European and North African regions (Browicz and Zohary, 1996; Kester and Gradziel, 1996; Xu et al., 2004) and it is also grown commercially around the world. Global almond production for 2018/19 is an estimated 1.4 million metric tons (USDA, 2018). Botanists observed over 30 species, subspecies with ecotype (Grasselly, 1976; Ladizinsky, 1999). Genetically, Almond is diploid, the chromosome number is 2n = 2x = 16 with a genome size approximately 246 Mb (Sánchez-Pérez et al., 2019). Almond is one of the vital plants that can grow under the rain-fed condition in Iraq, particularly in the Kurdistan region. Therefore, it is important to know the adaptation of this tree that able to tolerate the biotic and abiotic stresses. Almond is a large-sized tree, with long generation time and also has a low level of variability in morphology traits (Casas et al., 1999; Sorkheh et al., 2007, 2009; Zeinalabedini et al., 2008; Bouhadida et al., 2009). To provide information about genetic relationships it is important to study the APPLIED ECOLOGY AND ENVIRONMENTAL RESEARCH 18(1):1739-1753. http://www.aloki.hu ● ISSN 1589 1623 (Print) ● ISSN1785 0037 (Online) DOI: http://dx.doi.org/10.15666/aeer/1801_17391753 © 2020, ALÖKI Kft., Budapest, Hungary Mahood - Hama-Salih: Characterization of genetic diversity and relationship in almond genotypes by RAPD and ISSR markers in Sulaimani governorate - 1740 - differences at the level of agronomic, morphological and biochemical traits (Khan et al., 2016). Genetic diversity is an important tool that breeders can use to detect and identify with differentiation all genotypes and also it is a useful tool to improve the chances of the selection of better segregates for various characters (Dwevedi and Lal, 2009). Using morphological traits to the identification of almond plants is restricted, because of their environmental variations. However, morphological traits, including seed length and kernel size normally can be used. In Kurdistan, Iraq, information about almond genotypes has been poorly recognized. Therefore, the modern molecular genetic tool can be applied to identify and characterize the relationships among them. However, several similar studies have been performed regarding genetically recognized cultivars and wild species of almond. Molecular markers have shown the vital role in crop breeding, particularly in genetic diversity research and gene bank. PCR-based DNA marker systems are generally used, including Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and more recently simple sequence repeats (SSRs) or microsatellites (Gupta et al., 2000). Since no molecular evidence was obtainable concerning the almond genotypes grown in Sulaimani region, therefore, the consciousness and conception of the genetic diversity in Sulaimani almond accessions are important for the implementation of degree addressed to their usages and preservations. The present study was aimed to estimate the genetic diversity with relatedness among the most important almond genotypes and for the first time their population structure in Sulaimani was developed for almond genotypes by using RAPD and ISSR markers. Materials and Methods Locations and plant material Five locations (Fig. 1, Table 1) were selected for this study with thirty-eight almond local genotype trees distributed, including Sharbazher (9), Mergapan (3), Qaradakh (5), Barznja (7), and Hawraman (14). Figure 1. Distribution of collection sites of study plant materials in Sulaimani governorate APPLIED ECOLOGY AND ENVIRONMENTAL RESEARCH 18(1):1739-1753. http://www.aloki.hu ● ISSN 1589 1623 (Print) ● ISSN1785 0037 (Online) DOI: http://dx.doi.org/10.15666/aeer/1801_17391753 © 2020, ALÖKI Kft., Budapest, Hungary Mahood - Hama-Salih: Characterization of genetic diversity and relationship in almond genotypes by RAPD and ISSR markers in Sulaimani governorate - 1741 - Table 1. Genotypes name, location, latitude and altitude Latitude Genotypes name Locations Altitude N° W° SH-G1 to SH-G9 Sharbazher 35°49ʹ30ʺ 45°18ʹ93ʺ 997.6 M-G1 to M-G3 Mergapan 35°48ʹ93ʺ 45°13ʹ47ʺ 1148 Q-G1 to Q-G5 Qaradagh 35°19ʹ29ʺ 45°19ʹ69ʺ 925.8 B-G1 to B-G7 Barznja 35°27ʹ69ʺ 45°42ʹ21ʺ 1154 H-G1 to H-G14 Hawraman 35°12ʹ38ʺ 46°07ʹ80ʺ 1402 Morphological study of seeds Almond nuts were collected from each genotype (Fig. 2) Impurities such as damaged or broken nut, dust and dirt have been eliminated. For each nut, weight and size dimensions (the three axial dimensions) including length, width, and thickness of the nuts were measured by using a digital calliper with an accuracy of 0.01mm. The nuts were cracked, then using a hammer and kernel weighted to calculate shelling percentage. Figure 2. Shows the different morphological shapes of all almond genotypes nut, (Scale 1:1.75) APPLIED ECOLOGY AND ENVIRONMENTAL RESEARCH 18(1):1739-1753. http://www.aloki.hu ● ISSN 1589 1623 (Print) ● ISSN1785 0037 (Online) DOI: http://dx.doi.org/10.15666/aeer/1801_17391753 © 2020, ALÖKI Kft., Budapest, Hungary Mahood - Hama-Salih: Characterization of genetic diversity and relationship in almond genotypes by RAPD and ISSR markers in Sulaimani governorate - 1742 - Genomic DNA extraction and purification The CTAB DNA extraction protocol with some modifications was used to isolate genomic DNA, according to (Tahir and Hama Karim, 2011). Briefly, 1 gram of fresh leaves was ground and frozen in liquid nitrogen and then 2 ml of CTAB buffer was added and incubated at 60ºC for 60 min. (1 M Tris HCl (pH 8.0), 5 M NaCl, 0.5 M EDTA and 2 g of CTAB (cetyltrimethyl ammonium bromide)), the DNA sample was precipitated with 0.08 volumes of ammonium acetate and 0.54 volumes of ice isopropanol, and then the DNA pellet was washed with 1 ml of ice-cold 70% ethanol and then the dried pellet was resuspended in 50-100 µl of deionized water. DNA was treated with RNase to remove RNA contamination. The DNA concentration was analyzed by electrophoresis using a 1.5% agarose gel. RAPD analysis Twenty primers were utilized in this work, (Table 2). The reaction mixture (20 μl) was prepared for 1 volume sample (1x) which is 10 μl of master mix buffer with (0.7 μl) primer (20 pmol/ μl), 4 μl genomic DNA (100 ng/ μl) and then the total volume was completed up to 20 μl by distilled water. Amplification was carried out in a thermo-cycler (Master cycler) for 36 cycles, each consisting of an initial denaturation step at 94ºC for 10 minutes, denaturation at 94ºC for 1 minute, annealing temperature at at 36ºC for 1 minute with extension step at 72ºC for 2 minutes and then final synthesis step of 10 minutes at 72ºC, Amplification products were separated on 1.5% agarose gel in 1X TAE (Tris base, acetic acid and EDTA) buffer. Gels were run at a constant voltage of 100V for 60 minutes, then imaged using a UV trans-illuminator. The image was captured by a digital imaging system. ISSR analysis Fifteen primers were utilized in this study, (Table 2). The reaction mixture (20 μl) was prepared for one volume sample (1x) which is 10 μl of master mix buffer with 0.7 μl primer 20 pmol/ μl, 4 μl genomic DNA (100 ng/ μl) and then the total volume was completed up to 20 μl by distilled water. Amplification was carried out in a thermo-cycler (Master cycler) for 36 cycles, each consisting of an initial denaturation step at 94ºC for ten minutes, denaturation at 94ºC for 1 minute, annealing temperature at 50ºC for 1 minute with extension step at 72ºC for 2 minutes and then final synthesis step of 10 minutes at 72ºC, Amplification products were separated on 1.5% agarose gel in 1X TAE (Tris base, acetic acid and EDTA) buffer.